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      Defective Survival of Naive CD8 + T Lymphocytes in the absence of β3 regulatory subunit of Ca v channels

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          Abstract

          Survival of T lymphocytes requires sustained Ca 2+ influx-dependent gene expression. The molecular mechanism, which governs sustained Ca 2+ influx in naive T lymphocytes, is unknown. Here we report an essential role for the β3 regulatory subunit of Ca v channels in the maintenance of naive CD8 + T cells. β3 deficiency resulted in a profound survival defect of CD8 + T cells. This defect correlated with depletion of the pore-forming subunit Ca v1.4 and attenuation of T cell receptor-mediated global Ca 2+ entry in the absence of β3 in CD8 + T cells. Ca v1.4 and β3 associated with T cell signaling machinery and Ca v1.4 localized in lipid rafts. Our data demonstrate a mechanism by which Ca 2+ entry is controlled by a Ca v1.4–β3 channel complex in T cells.

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          Most cited references40

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          Orai1 is an essential pore subunit of the CRAC channel.

          Stimulation of immune cells causes depletion of Ca2+ from endoplasmic reticulum (ER) stores, thereby triggering sustained Ca2+ entry through store-operated Ca2+ release-activated Ca2+ (CRAC) channels, an essential signal for lymphocyte activation and proliferation. Recent evidence indicates that activation of CRAC current is initiated by STIM proteins, which sense ER Ca2+ levels through an EF-hand located in the ER lumen and relocalize upon store depletion into puncta closely associated with the plasma membrane. We and others recently identified Drosophila Orai and human Orai1 (also called TMEM142A) as critical components of store-operated Ca2+ entry downstream of STIM. Combined overexpression of Orai and Stim in Drosophila cells, or Orai1 and STIM1 in mammalian cells, leads to a marked increase in CRAC current. However, these experiments did not establish whether Orai is an essential intracellular link between STIM and the CRAC channel, an accessory protein in the plasma membrane, or an actual pore subunit. Here we show that Orai1 is a plasma membrane protein, and that CRAC channel function is sensitive to mutation of two conserved acidic residues in the transmembrane segments. E106D and E190Q substitutions in transmembrane helices 1 and 3, respectively, diminish Ca2+ influx, increase current carried by monovalent cations, and render the channel permeable to Cs+. These changes in ion selectivity provide strong evidence that Orai1 is a pore subunit of the CRAC channel.
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            Genome-wide RNAi screen of Ca(2+) influx identifies genes that regulate Ca(2+) release-activated Ca(2+) channel activity.

            Recent studies by our group and others demonstrated a required and conserved role of Stim in store-operated Ca(2+) influx and Ca(2+) release-activated Ca(2+) (CRAC) channel activity. By using an unbiased genome-wide RNA interference screen in Drosophila S2 cells, we now identify 75 hits that strongly inhibited Ca(2+) influx upon store emptying by thapsigargin. Among these hits are 11 predicted transmembrane proteins, including Stim, and one, olf186-F, that upon RNA interference-mediated knockdown exhibited a profound reduction of thapsigargin-evoked Ca(2+) entry and CRAC current, and upon overexpression a 3-fold augmentation of CRAC current. CRAC currents were further increased to 8-fold higher than control and developed more rapidly when olf186-F was cotransfected with Stim. olf186-F is a member of a highly conserved family of four-transmembrane spanning proteins with homologs from Caenorhabditis elegans to human. The endoplasmic reticulum (ER) Ca(2+) pump sarco-/ER calcium ATPase (SERCA) and the single transmembrane-soluble N-ethylmaleimide-sensitive (NSF) attachment receptor (SNARE) protein Syntaxin5 also were required for CRAC channel activity, consistent with a signaling pathway in which Stim senses Ca(2+) depletion within the ER, translocates to the plasma membrane, and interacts with olf186-F to trigger CRAC channel activity.
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              Suppression of IL7Ralpha transcription by IL-7 and other prosurvival cytokines: a novel mechanism for maximizing IL-7-dependent T cell survival.

              Survival of naive T cells is dependent upon IL-7, which is present in vivo in limiting amounts with the result that naive T cells must compete for IL-7-mediated survival signals. It would seem imperative during T cell homeostasis that limiting IL-7 be shared by the greatest possible number of T cells. We now describe a novel regulatory mechanism that specifically suppresses IL7Ralpha transcription in response to IL-7 and other prosurvival cytokines (IL-2, IL-4, IL-6, and IL-15). Consequently, IL7R expression is reduced on T cells that have received cytokine-mediated survival signals so they do not compete with unsignaled T cells for remaining IL-7. Interestingly, cytokine-mediated suppression of IL7Ralpha transcription involves different molecular mechanisms in CD4+ and CD8+ T cells, as CD8+ T cells utilize the transcriptional repressor GFI1 while CD4+ T cells do not. We suggest that this homeostatic regulatory mechanism promotes survival of the maximum possible number of T cells for the amount of IL-7 available.
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                Author and article information

                Journal
                100941354
                21750
                Nat Immunol
                Nature immunology
                1529-2908
                1529-2916
                24 August 2009
                18 October 2009
                December 2009
                1 June 2010
                : 10
                : 12
                : 1275-1282
                Affiliations
                [1 ]Department of Immunobiology, Yale University School of Medicine, New Haven, CT 06510, USA.
                [2 ]Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT 06510, USA.
                [3 ]Université Cadi Ayyad, Faculté polydisciplinaire Safi, Sidi Bouzid, B.P. 4162, 46000 Safi, Morocco.
                [4 ]Institute for Experimental and Clinical Pharmacology and Toxicology, University of Saarland, 66421 Homburg, Germany.
                [5 ]Hotchkiss Brain Institute, University of Calgary, Calgary, Canada.
                Author notes

                Author contributions

                M.K.J. conceptualized, designed and did all experiments except calcium imaging and wrote the paper; A.B. performed & analyzed calcium-imaging experiments; A.B., M.M., J.E.M., M.F., and V.F. contributed reagents; R.A.F. provided overall directions, supervised the project and wrote the paper.

                Correspondence should be addressed to R.A.F. ( richard.flavell@ 123456yale.edu )
                Article
                hhmipa140662
                10.1038/ni.1793
                2785134
                19838200
                08e1e8a0-219d-4cad-9ea0-4753f6723bee

                Users may view, print, copy, download and text and data- mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: http://www.nature.com/authors/editorial_policies/license.html#terms

                History
                Funding
                Funded by: Howard Hughes Medical Institute
                Award ID: ||HHMI_
                Categories
                Article

                Immunology
                apoptosis,cav channel,t lymphocyte,cacnb3,calcium
                Immunology
                apoptosis, cav channel, t lymphocyte, cacnb3, calcium

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